Synergistic antibiotics

ABSTRACT

Rifamycines or their semisynthetic derivatives, in combination with other antibiotics selected from oligosaccharides, tetracyclines, amphenicols, macrolides or peptides exhibit synergistic effects against pathogens.

D United States Patent [191 [111 3,725,543 Konopka et al. 1 Apr. 3, 1973[54] SYNERGISTIC ANTIBIOTICS [58] Field of Search "424/1 14 [75]Inventors: Edward Alexander Konopka, Murray Hill, N.J.; Justus Melchior[56] References cued Geller, Richer], Switzerland [731 Assignee gf'Ardsleyi R. Virchow et al., Dtsch. Med. Wscar. 92Jq., 1967,

a es 2217-2220 P g [22] Filed: Dec. 8, 1971 Primary Examiner.lerome D.Goldberg [211 Appl' 206l85 Att0rneyJoseph G. Kolodny et al.

Related 0.8. Application Data [57] ABSTRACT [63] Continuation-impart ofSer. No. 81,112, Oct. 20,

1970, which is a continuation-in-part of Ser, No, Rifamycines or theirsemisynthetic derivatives, in 13,788, Feb. 24, 1970, abandoned, which isa concombination with other antibiotics selected from -P of 309,967,March 24, oligosaccharides, tetracyclines, amphenicols, macro- 1969,3v644616- lides or peptides exhibit synergistic effects againstpathogens. [52] U.S.Cl ..424/1l4 [51] Int. Cl. ..A61k 21/00 6 Claims, NoDrawings SYNERGISTIC ANTIBIOTICS CROSS-REFERENCES TO RELATEDAPPLICATIONS This is a continuation-in-part of application, Ser. No. 5

81,112, filed Oct. 15, 1970, which in turn is a continuation-in-part ofapplication Ser. No. 13,788, filed Feb. 24, 1970 (now abandoned), whichin turn is a continuation-in-part of application, Ser. No. 809,967,filed Mar. 24, 1969 now U.S. Pat. No. 3,644,616. 1

SUMMARY OF THE INVENTION The present invention concerns and has for itsobject the provision of new pharmaceutical or veterinary 1 compositions,feedstuffs or feed additives comprising synergistic combinations of 1)rifamycines or their semisynthetic derivatives, with 2) antibioticsselected from oligosaccharides, tetracyclines, amphenicols, macrolidesor peptides, as well as ,of methods for the preparation and applicationof these products, which are useful antibacterial agents or growthpromoters.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The rifamycines of the newcompositions and feed preparations are known and represent especiallythe rifamycines B, SV, S, 0, AG or X, preferably their semisyntheticderivatives, e.g. rifamide, rifazine or advantageously rifampicin. Saidcomponent is described, inter alia, in I1 Farmaco, Ed. Sci. 16, 755 and766 (1961), 21, 68 (1966) and 22, 307 (1967); J. Med. Chem. 7, 596(1964), 8 790 (1965) and 11, 936 (1968); Antimicrobial Agents andChemotherapy (Am. Soc. Microbiol.) 1965, p. 765 or 1967, p. 699, theReport of the 5th lnternatl. Congr. of Chemotherapy, 1967; French Pat.Nos. 1,434,532, 1,457,435 and 5518M, Belgian Patent Nos. 654,209 and685,886, South African Patent 68/0903 and U.S. Pat. No. 3,349,082.

The antibiotics used are also known and are represented by the group ofnatural or semisynthetic a) saccharides,such as streptomycin A or B oroxystreptomycin, or their semisynthetic derivatives, e.g.dihydrostreptomycin or -oxystreptomycin, or preferably the highersaccharides, especially the trisaccharides, e.g. kanamycin (A, B or C),glebomycin or gentamycin; also the tetrasaccharides, e.g. neomycin orparomycin, b) tetracyclines, e.g. tetracycline, chlortetracylcine,demethylchlortetracycline, doxy-cycline, lymecylcine, meclocycline,methacycline, minocycline, nitrocycline, oxytetracycline,rolitetracycline or sancycline, c) amphenicols, e.g. chloramphenicol,azidoamphenicol, thiamphenicol or fluophenicol, d) macrolides, e.g.carbomycin (A or B), erythromycin, oleandomycin, pikromycin, spiramycinor troleandomycin, or e) piptides, e.g. amphomycin, bacitracin,colistin, gramicidin, lincomycin, novobiocin, polymixin, ristocetin,tyrocidin (A, B or C), vancomycin or viomycin, which are described,inter alia, in Erhart-Ruschig, Arzneimittel I1, 1571 et seq. (VerlagChemie, Weinheim 1968). Said book also describes the rifamycines on page1517 et seq.

The compositions and feed preparations according to the inventioncontain an effective amount of the rifamycines and other antibiotics ina ratio between about 1:10 and 10:1, preferably between about 1:5 and5:1, especially between about 1:2 and 2:1, and the usual amount ofconventional excipients or' extenders, whereby the total amount of bothantibiotics can be less than that used in the known preparations of thecomponents.

The antibacterial effects of the new combinations can be demonstratedeither in vitro or in vivo tests. For example, the growth dynamics ofGram-negative or positive bacteria can be estimated in media whichcontain, besides the nutrients,

a. no antibiotic (control (1)).

b. the rifamycines (A) or other antibiotics (B) alone,

0. both components (A B).

The single amounts of A and B used according to c) can be half of thoseused according to b). In vivo tests are performed with various testanimals, advantageously with mammals, such as mice, which are challengedintravenously or intraperitoneally with a lethal or sublethal amount ofpathogens, whereupon the compositions of the invention are administeredeither enterally or parenterally, e.g. orally or subcutaneously. Theireffect can either be determined by the curing (or survival) rate ofinfected animals or by the recovery of viable pathogens from theirorganism, for example, from particular organs, such as kidneys.

Surprisingly, it has been found that the growth dynamics of pathogensmore particularly of the antibiotically less tractable Gram-negativebacteria, such as the Aerobacter, Brucella, Escherichia, Klebsiella,Malleomyces, Neisseria, Pasteurella, Proteus, Pseudomonas, Salmonella,Shigella and Vibrio strains, as well as those of Gram-positive bacteria,such as Actinomyces, Clostridia, Corynebacteria, Diplococci,Mycobacteria, Staphylococci or Streptococci, are beneficially altered bythe compositions of the invention. For example, said in vitro growthdynamics according to items a), b) and c) can be depicted as follows:

Number not a) surviving bacteria Time t 20 (hours) These functionsf(n,t) depicting said growth dynamics indicate that the antibacterialeffect of a combination according to the invention is bigger than thatobtainable with the same amount of the components.

Accordingly, the new compositions and feed preparations are superior tothose of the presently used components, since lesser doses can beapplied. Moreover, a suppression of resistance development can beachieved. For example, pathogens attacked with the rifamycines or theirsemisynthetic derivatives, seem to be more sensitive to the otherantibiotics mentioned above under item 2), especially penicillines,cephalosporines, streptomycines, kanamycines or'gentamycines.

Particularly useful are pharmaceutical or veterinary compositions, aswell as feedstuffs and feed additives, comprising an effective amountof 1. a rifamycin selected from the group consisting of rifamycin (AG,B, O, S, SV or X),rifamide, rifampicin and rifazine or a therapeuticallyuseful salt thereof and another antibiotic selected from the groupconsisting of kanamycin (A, B or C), glebomycin or gentamycin; neomycinor paromycin; tetracycline, chlortetracycline,demethylchlortetracycline, doxycycline, lymecylcine, meclocycline,methacycline, minocycline, nitrocycline, oxytetracycline,rolitetracycline or sancycline; chloramphenicol, azidoamphenicol,thiamphenicol or fluophenicol; carbomycin (A or B), erythromycin,oleandomycin, pikromycin, spiramycin or troleandomycin; amphomycin,bacitracin, colistin, gramicidin, lincomycin, novobiocin, polymixin,ristocetin, tyrocidin (A, B or C), vancornycin or viomycin; or atherapeutically useful salt thereof.

Especially valuable are compositions and feed preparations containing apharmacologically effective amount of 1. rifamycin SV, rifamide,rifampicin or rifazine, or a therapeutically useful salt thereof andkanamycin, gentamycin or neomycin; tetracycline, chlortetracycline,demethylchlortetracycline, doxycycline, methacycline, oxytetracycline orrolitetracycline; chloramphenicol; erythromycin, oleandomycin ortroleandomycin; bacitracin, colistin, gramicidin, novobiocin, polymixinor vancomycin; or a therapeutically useful salt thereof.

Outstanding are compositions containing an antibiotically effectiveamount of 1. rifampicin or a therapeutically useful salt thereof and 2.either a) kanamycin, gentamycin or neomycin, or

b) tetracycline, chlortetracycline or oxytetracycline, or c)chloramphenicol or d) colistin; or a therapeutically useful saltthereof.

Said compositions and feed preparations advantageously contain but oneof the above antibiotics mentioned under items 1) and 2) but can containmore than one of each, for example, more than one of the antibioticslisted under item 2). The preferred proportions of the activeingredients of said compositions and feed preparations range betweenabout 1:5 and 5:1, advantageously between about 1:2 and 2:1. In additionto the active ingredients, they contain the usual amount of conventionalexcipients or extenders.

The pharmaceutical or veterinary compositions according to the inventioncontain both of said antibiotics mentioned under 1) and 2) in about thesame or a lesser amount than that used in conventional compositions ofthe components, in conjunction or admixture with excipients suitable foreither enteral, parenteral or topical application. Preferred are tabletsand gelatin capsules comprising the active ingredients together with a)diluents, e.g. lactose, dextrose, sucrose, mannitol, sorbitol, celluloseand/or glycine, b) lubricants, e.g. silica, talcum, stearic acid, itsmagnesium or calcium salt and/0r polyethyleneglycol, for tablets also c)binders, e.g. magnesium aluminum silicate, starch paste, gelatin,tragacanth, methylcellulose, sodium carboxymethylcellulose and/orpolyvinylpyrrolidone, if desired, (1) disintegrants, e.g. starches,agar, alginic acid or its sodium salt, enzymes of the binders oreffervescent mixtures and/or e) adsorbents, colorants, flavors andsweeteners. Injectable compositions are preferably aqueous isotonicsolutions or suspensions, and suppositories or ointments areadvantageously fatty emulsions or suspensions. They may be sterilizedand/or contain adjuvants, such as preserving, stabilizing, wetting oremulsifying agents, solution promoters, salts for regulating the osmoticpressure and/or buffers. Said pharmaceutical compositions may alsocontain other therapeutically valuable substances. They are preparedaccording to conventional mixing, granulating or coating methodsrespectively and contain about 0.1 to percent, preferably about 1 to 50percent of the active ingredients listed under 1 and 2).

The feedstuffs or additives for feed or drinking water contain both ofsaid antibiotics also in about the same or a lesser amount as that usedin conventional feedstuffs or additives of the components, which areintended to promote the growth and feed efficiency of domestic animals.Said feedstuffs or additives also contain the conventional extenders,diluents and/or nutrients, such as sucrose, glucose, molasses,fermentation residues, corn meal, ground and rolled oats, wheat shortsand middlings, meat scrap, oil cake, soybean and fish meal, alfalfa,clover or grass clippings and the like, mineral supplements, such asbone meal, calcium carbonate, iodized salt and the like, vitamins, suchas vitamins A, B, C and D, and other suitable substances, such aspreservants, e.g. benzoic acid. The feedstuffs contain the activeingredients advantageously in the dosage range, for example, betweenabout 0.00001 and 0.01 percent, whereas the additives may consist of thepure substances, when used, for example, for the drinking water, butusually contain between about 1 and 75 percent thereof.

The following examples illustrate the invention and are not to beconstrued as being limitations thereon. Temperatures are given indegrees Centigrade and all parts wherever given are parts by weight.

EXAMPLE 1 Conventional test tubes are filled with 10 ml of conventionaltrypticase broth a) alone, or such containing b) rifampicin or the otherantibiotics, or c) the combinations thereof, in a concentration betweenabout 5-20 ug/ml.

Hereupon 0.1 ml of a freshly grown and standardized stock culture ofEscherichia coli is added to said 10 ml broth, so that it containsapproximately 10 organisms per ml. After 20 hours incubation at 37, 1 mlsamples are removed and the number of viable cells therein determinedaccording to the plate dilution method. It provides from a number ofbacterial cells in a properly diluted specimen an equal number ofvisible colonies, which can be counted.

The following results are obtained:

Antibiotic Concentration No. of Cells After ag/ml 0 and 20 hours none 01.7.10 1.7.10 rifampicin (A) 5 1.7.10 1.0.10 streptomycin (B) 10 1.6.101.0.10 A+B 5+l0 1.8.10 6.8.10

EXAMPLE 2 According to the method described in Example I,

the following results are obtained with Proteus mirabilis:

Antibiotic Concentration No. of Cells After Lg/ml and 20 hours none 01.8.10 1.8.10 rifampicin (A) 2.4.10 1.2.10" 2.810 7.8.10 2.1.10 [010streptomycin (B) 5 1.6.10 1.5.10 10 2.0.10 1.2.10 20 1.5.10 1.1.10" A+B5+5 2.3.10 7.5.10 5+ 10 2.2.10 1.5.10 10+5 2.1.10 2.3.10 l0+l0 1.5.101.5.10

EXAMPLE 3 According to the method described in Example 1, the followingresults are obtained with rifampicin-resistant cells of E. coli:

Concentration No. of Cells After Antibiotic mg/ml 0 and 24 hours none 03.6.10 1.3.10 rifampicin (A) 20 3.3.10 1.2.10 streptomycin (B) 20 3.6.109.7.10 A+B 20+20 3.8.10 9.0.10

EXAMPLE 4 White laboratory mice are infected by the i.p. application ofa sufficient amount of Escherichia coli pathogens suspended in saline,which cause death of 90-l00 percent of untreated control animals within48 hours. About 30-60 minutes after infection, groups of 10 mice eachare treated with the antibiotics shown in the table below, either aloneor in combination, which antibiotics are administered only once in theform of aqueous solutions or suspensions orally. Said in vivaexperiments are terminated at the 10th to 14th day after infection andthe survivors counted. Most of the experiments were carried out twiceand the average value estimated.

The following results were obtained:

Antibiotic Dose in mglkg Survivors rifampicin (A) 50 tetracycline (B) 2550 A B 25 25 100 A 25 50 B 10 0 A B 25 10 85 A 10 0 B 25 50 A B 10 25 75EXAMPLE 5 Groups of 10 white laboratory mice are infected by theintraperitoneal application of a suspension containing 10 cells ofPseudomonas aeruginosa. This infecting dose causes 90-100 percentmortality in the untreated controls within 48 hours.

Medication is applied only once within one hour following infection, bythe subcutaneous administration of the antibiotics listed below. Aqueoussolutions or suspensions of the individual and combined antibiotics arefreshly prepared each day and are allowed to remain at room temperaturefor 1 hour prior to administration.

The mice tested are observed for 14 days following infection and thesurvivors counted. Several experiments are repeated and the averagevalue estimated. The following results were obtained:

A Rifampicin, B Streptomycin, C Gentamycin, D Kanamycin,

E Neomycin, F Oxytetracyclin, Chloramphenicol, H =sodium colistimethate.

Antibiotic Dose mg/ltg Percent Survivors none 0 0 A 20 0 B 20 30 A B 2020 90 A 20 0 B 10 20 A B 20 +10 none 0 10 A 20 10 C 5 10 A C 20 5 100 A20 10 C 2.5 10 A C 20 2.5 A 10 0 C 5 10 A C 10 5 50 A l0 10 C 2.5 10 A C10 2.5 30 none 0 0 A 20 0 D 50 45 A D 20 50 80 A 10 0 D 50 45 A D 10 5080 none 0 0 A 20 0 E 20 70 A E 20 20 100 A 20 0 E 10 65 A E 20 +10 A 200 E 5 10 A E 20 5 70 A 10 0 E 20 70 A E 10 20 95 A 10 0 E 10 65 A E 10+10 90 A 0 0 E 5 10 A E 10 5 30 none 0 0 A 20 0 F 200 20 A F 20 200 90 A20 0 F 150 20 A F 20+ 150 90 A 20 0 F 0 A F 20 +100 4 A 10 0 F 200 20 AF 10 200 65 A 10 0 F 20 A F 10 +150 70 A 10 0 F 100 0 A F 10 100 40 none0 0 A 20 0 G 200 0 A G 20 200 90 A 20 0 G 150 0 A G 20 150 30 none 0 0 A20 0 H 20 10 A H 20 20 100 A 20 0 H 10 0 A H 20 +10 50 A 10 0 1-1 20 10A+H 10+20 100 A 10 H 0 A+H 10+ 10 EXAMPLE 6 Preparation of 1,000capsules each containing 300 mg of the active ingredients:

Formula: Rifampicin 150 g Gentamycin 150 g Talcum 36 g Corn starch 24 gMagnesium stearate 16 g Lactose 4 g 380 g PROCEDURE All powders arepassed through a screen with an opening of 0.6 mm and mixed thoroughly.0.5 ml hard gelatine capsules are filled with 380 mg of said mixture,using a capsule filling machine.

EXAMPLE 7 Preparation of 1,000 capsules each containing 150 mg of theactive ingredients:

Formula: rifampicin 75 g kanamycin 75 g ethyl cellulose 3 g stearic acid3 g I56 g PREPARATION The ethyl cellulose and stearic acid are dissolvedin 120 ml methylene chloride, the antibiotics are added and the masspassed through a sieve with 0.6 mm openings at a temperature of about 40whereby the methylene chloride evaporates. 156 mg of the granulateobtained are filled into 0.5 ml hard gelatine capsules using a capsulefilling machine.

EXAMPLE 8 Preparation of a poultry feed containing 0.005 percent of theactive ingredients:

Premix: rifampicin g chlortetracycline 25 g confectioners su ar 50 gsoybean feed, so vent extracted 275 g 375 g Feed Formula: Pounds Cornmeal 1103.0 So bean meal, 44% protein 660.0 Al alfa meal 30.0

Dicalcium phos hate Limestone me Salt Fish meal, 60% protein Stabilizedfat 5 Dried whey Manganese sulfate Zinc oxide d,1-methionine Vitaminpremix The antibiotics and sugar are mixed thoroughly, screened througha sieve with 0.6 mm openings and blended with the soybean feed. Thepremix is then added to the feed in such amount as to obtain saidconcentration and the whole is homogenized in a horizontal drum mixer.

According to the method described in Examples 6-8, capsules or feedmixtures are prepared containing equieffective amounts of the otherantibiotics hereinbefore described or exemplified.

We claim:

1. An antibiotic pharmaceutical or veterinary composition, consisting ofan antibiotically effective amount of 1) rifampicin, 2) oxytetracycline,or a therapeutically useful salt of either component, wherein theproportions of the rifampicin to the oxytetracycline is 1:75 to 1:20 and3) a pharmaceutical excipient suitable for enteral application.

2. A composition as claimed in claim 1, wherein the pharmaceuticalexcipinet, and the component 3) is a pharmaceutical capsule excipient.

3. A composition as claimed in claim 1, containing between about 0.1 to75 percent of the components 1) and 2) in the pharmaceutical excipient3).

4. An antibiotic feedstuff or feed additive consisting of a growthpromoting an amount of 1) rifampicin, 2) oxytetracycline, or atherapeutically useful salt of either component, wherein the proportionsof the rifampicin to the oxytetracycline is 1:7.5 to 1:20 and 3) anextender, diluent or nutrient.

5. A feedstuff as claimed in claim 4, containing between about 0.00001and 0.01 percent of the components 1) and 2) in a nutrient.

6. A feed additive as claimed in claim 4, containing between about 1 and75 percent of the components 1) and 2) in an extender.

l I l t

2. A composition as claimed in claim 1, wherein the pharmaceuticalexcipinet, and the component 3) is a pharmaceutical capsule excipient.3. A composition as claimed in claim 1, containing between about 0.1 to75 percent of the components 1) and 2) in the pharmaceutical excipient3).
 4. An antibiotic feedstuff or feed additive consisting of a growthpromoting an amount of 1) rifampicin, 2) oxytetracycline, or atherapeutically useful salt of either component, wherein the proportionsof the rifampicin to the oxytetracycline is 1:7.5 to 1:20 and 3) anextender, diluent or nutrient.
 5. A feedstuff as claimed in claim 4,containing between about 0.00001 and 0.01 percent of the compOnents 1)and 2) in a nutrient.
 6. A feed additive as claimed in claim 4,containing between about 1 and 75 percent of the components 1) and 2) inan extender.